CRISPR-Based Diagnostic Assay for Point-of-Care Detection of Methicillin-Resistant Staphylococcus aureus in patients with Bloodstream Infections

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of mortality in critically ill patients with bloodstream infections (BSIs). Timely diagnosis and early initiation of guideline-concordant antimicrobial therapy are independently associated with improved survival and reduced complication rates in patients with MRSA BSIs. However, current blood culture-based detection of MRSA requires 3-7 days for definitive identification and antimicrobial susceptibility reporting, entails numerous manual steps, and frequently delays the transition from empirical to pathogen-directed therapy, thereby affecting treatment outcomes and patient prognosis. Here, we present a highly sensitive and specific CRISPR-based diagnostic platform that shortens the MRSA detection time from >70 h to ∼2 h-enabling true "sample-to-result" testing directly from whole blood at the point of care. This study develops a one-step RPA-CRISPR assay targeting the S. aureus nucA and mecA genes, with 10 copies/reaction sensitivity and no cross-reactivity. By integrating vancomycin-conjugated magnetic nanoparticles, a herringbone microfluidic chip, and a miniaturized wireless analyzer, the platform performs on-chip S. aureus capture, lysis-mediated DNA release, and dual-gene detection directly from whole blood. In a prospective, multicenter clinical evaluation of 350 hospitalized patients with suspected bacteremia, the HELP platform identified all 20 S. aureus-positive cases (including 8 MRSA-positive cases), demonstrating 100% positive percent agreement with blood culture. By delivering definitive MRSA identification in under 2 h, HELP markedly accelerates clinical decision-making, facilitates the timely initiation of targeted antimicrobial therapy, and holds strong potential for scalable deployment in primary care and resource-constrained settings.